A Systematic Bioinformatics Approach for Mapping the Minimal Set of a Viral Peptidome.

Sequence changes in viral genomes generate protein sequence diversity that enables viruses to evade the host immune system, hindering the development of effective preventive and therapeutic interventions. The massive proliferation of sequence data provides unprecedented opportunities to study viral adaptation and evolution. An alignment-free approach removes various restrictions posed by an alignment-dependent approach for studying sequence diversity. The publicly available tool, UNIQmin, offers an alignment-free approach for studying viral sequence diversity at any given rank of taxonomy lineage and is big data ready. The tool performs an exhaustive search to determine the minimal set of sequences required to capture the peptidome diversity within a given dataset. This compression is possible through the removal of identical sequences and unique sequences that do not contribute effectively to the peptidome diversity pool. Herein, we describe a detailed four-part protocol utilizing UNIQmin to generate the minimal set for the purpose of viral diversity analyses, alignment-free at any rank of the taxonomy lineage, using the recent global public health threat Monkeypox virus (MPX) sequence data as a case study. The protocol enables a systematic bioinformatics approach to study sequence diversity across taxonomic lineages, which is crucial for our future preparedness against viral epidemics. This is particularly important when data are abundant, freely available, and alignment is not an option. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Tool installation and input file preparation Basic Protocol 2: Generation of a minimal set of sequences for a given dataset Basic Protocol 3: Comparative minimal set analysis across taxonomic lineage ranks Basic Protocol 4: Factors affecting the minimal set of sequences.

dgfr: an R package to assess sequence diversity of gene families.

BACKGROUND: Gene families are groups of homologous genes that often have similar biological functions. These families are formed by gene duplication events throughout evolution, resulting in multiple copies of an ancestral gene. Over time, these copies can acquire mutations and structural variations, resulting in members that may vary in size, motif ordering and sequence. Multigene families have been described in a broad range of organisms, from single-celled bacteria to complex multicellular organisms, and have been linked to an array of phenomena, such as host-pathogen interactions, immune evasion and embryonic development. Despite the importance of gene families, few approaches have been developed for estimating and graphically visualizing their diversity patterns and expression profiles in genome-wide studies. RESULTS: Here, we introduce an R package named dgfr, which estimates and enables the visualization of sequence divergence within gene families, as well as the visualization of secondary data such as gene expression. The package takes as input a multi-fasta file containing the coding sequences (CDS) or amino acid sequences from a multigene family, performs a pairwise alignment among all sequences, and estimates their distance, which is subjected to dimension reduction, optimal cluster determination, and gene assignment to each cluster. The result is a dataset that allows for the visualization of sequence divergence and expression within the gene family, an approximation of the number of clusters present in the family. CONCLUSIONS: dgfr provides a way to estimate and study the diversity of gene families, as well as visualize the dispersion and secondary profile of the sequences. The dgfr package is available at https://github.com/lailaviana/dgfr under the GPL-3 license.

Prevalence and sequence diversity of Balantioides coli in pigs in Xinjiang, China.

Balantioides coli is a common intestinal parasitic protozoan in pigs. In the present study, 801 fecal samples of pigs from seven farms in Xinjiang were analyzed based on the ITS1-5.8S rRNA-ITS2 gene. The prevalence of B. coli was 4.2% (34/801), with the highest prevalence of 18.9% (18/95) occurring in Alaer, Xinjiang. B. coli was detected in all age groups (pre-weaned pigs, post-weaned pigs, fattening pigs and sows), with the highest rate in fatteners (6.9%, 9/129) and the lowest (1.2%, 2/169) in pre-weaned pigs. Significant differences (P = 0.000) were found among sampling sites but not among age groups (P = 0.084). Sequence analysis indicated that 34 sequence variants, including sequence type A (n = 11) and sequence type B (n = 23), occurred in all age groups. In this study, the existence of sequence type A suggested that B. coli poses a potential threat to human health. More studies are needed to better understand the distributions and public health significance of B. coli in China.

Sequence, structure and functionality of pectin methylesterases and their use in sustainable carbohydrate bioproducts: A review.

Pectin methylesterases (PMEs) are enzymes that play a critical role in modifying pectins, a class of complex polysaccharides in plant cell walls. These enzymes catalyze the removal of methyl ester groups from pectins, resulting in a change in the degree of esterification and consequently, the physicochemical properties of the polymers. PMEs are found in various plant tissues and organs, and their activity is tightly regulated in response to developmental and environmental factors. In addition to the biochemical modification of pectins, PMEs have been implicated in various biological processes, including fruit ripening, defense against pathogens, and cell wall remodelling. This review presents updated information on PMEs, including their sources, sequences and structural diversity, biochemical properties and function in plant development. The article also explores the mechanism of PME action and the factors influencing enzyme activity. In addition, the review highlights the potential applications of PMEs in various industrial sectors related to biomass exploitation, food, and textile industries, with a focus on development of bioproducts based on eco-friendly and efficient industrial processes.

Sequence diversity of hepatitis D virus in Mongolia.

Introduction: The Hepatitis Delta Virus (HDV) is a defective, single-stranded RNA virusoid encoding for a single protein, the Hepatitis Delta Antigen (HDAg), which requires the hepatitis B virus (HBV) envelope protein (HBsAg) for its transmission. Currently, hepatitis D is the most aggressive form of viral hepatitis and treatment options are limited. Worldwide 12 million people are chronically infected with HDV being at high risk for progression to cirrhosis and development of liver cancer. Objectives: Although it is well established that Mongolia is the country with the highest prevalence of HDV infections, the information on the molecular epidemiology and factors contributing to HDV sequence diversity are largely unclear. The aim of the study was to characterize the sequence diversity of HDV in rural areas from Mongolia and to determine the extent of HLA class I-associated selection pressure. Patients and methods: From the HepMongolia cohort from rural areas in Mongolia, 451 HBsAg-positive individuals were selected and anti-HDV, HDV-RNA and the sequence of the large HDAg was determined. For all individuals the HLA class I locus was genotyped. Residues under selection pressure in the presence of individual HLA class I types were identified with the recently published analysis tool HAMdetector. Results: Of 431 HBsAg positive patients, 281 were anti-HDV positive (65%), and HDV-RNA could be detected in 207 of 281 (74%) of patients. The complete large HDAg was successfully sequenced from 131 samples. Phylogenetic analysis revealed that all Mongolian HDV isolates belong to genotype 1, however, they separate into several different clusters without clear regional association. In turn, from phylogeny there is strong evidence for recent local transmission events. Importantly, we found multiple residues with strong support for HLA class I-associated selection pressure consistent with a functional CD8+ T cell response directed against HDV. Conclusion: HDV isolates from Mongolia are highly diverse. The molecular epidemiology suggests circulation of multiple subtypes and provides evidence for ongoing recent transmissions.

DNA barcoding of Japanese earwig species (Insecta, Dermaptera), with sequence diversity analyses of three species of Anisolabididae.

Dermaptera is a polyneopteran insect order that includes more than 2,000 described species, commonly known as earwigs, that mainly inhabit tropical, subtropical and warm temperate regions. Although 40 species have been found in Japan, their distribution and habitat preferences have remained ambiguous due to sample misidentification, particularly amongst immature specimens. To overcome this problem, we sequenced and analysed the DNA barcoding region of the mitochondrial cytochrome oxidase I gene (cox1) of dermapteran species recorded from Japan. Including publicly available data, 72.5% of known Japanese dermapteran species were subjected to molecular identification. We extensively sampled three wingless species of subfamily Anisolabidinae (Anisolabididae): Anisolabismaritima, Anisolabellamarginalis and Euborelliapallipes. Although these species exhibit similar habitat preferences as semi-synanthropes, A.maritima, a cosmopolitan species with the highest affinity to seashore, had significantly higher sequence diversity than the latter two species, which are considered endemic to East Asia. A similar trend was observed for (at least partly) winged cosmopolitan species of other families. Introgression with the congener Anisolabisseirokui is also suggested for A.maritima. Possible causes of the varying levels of sequence diversity are discussed.

Corrigendum: Chloroplast genome sequencing, comparative analysis, and discovery of unique cytoplasmic variants in pomegranate (Punica granatum L.).

[This corrects the article DOI: 10.3389/fgene.2021.704075.].

Uncovering the promiscuous activity of IL-6 proteins: A multi-dimensional analysis of phylogeny, classification and residue conservation.

The IL-6 family of cytokines, known for their pleiotropic behavior, share binding to the gp130 receptor for signal transduction with the necessity to bind other receptors. Leukemia inhibitory factor receptor is triggered by the IL-6 family proteins: leukemia inhibitory factor (LIF), oncostatin-M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine factor 1 (CLCF1). Besides the conserved binding sites to the receptor, not much is known in terms of the diversity and characteristics of these proteins in different organisms. Herein, we describe the sequence analysis of LIF, OSM, and CT-1 from several organisms, and m17, a LIF ortholog found in fishes, regarding its phylogenetics, intrinsic properties, and the impact of conserved residues on structural features. Sequences were identified in seven classes of vertebrates, showing high conservation values in binding site III, but protein-dependent results on binding site II. GRAVY, isoelectric point, and molecular weight parameters were relevant to differentiate classes in each protein and to enable, for the first time and with high fidelity, the prediction of both organism class and protein type just using machine learning approaches. OSM sequences from primates showed an increased BC loop when compared to the remaining mammals, which could influence binding to OSM receptor and tune signaling pathways. Overall, this study highlights the potential of sequence diversity analysis to understand IL-6 cytokine family evolution, showing the conservation of function-related motifs and evolution of class and protein-dependent characteristics. Our results could impact future medical treatment of disorders associated with imbalances in these cytokines.

Comparative genomic analysis and phylogeny of NAC25 gene from cultivated and wild Coffea species.

Coffee is a high value agricultural commodity grown in about 80 countries. Sustainable coffee cultivation is hampered by multiple biotic and abiotic stress conditions predominantly driven by climate change. The NAC proteins are plants specific transcription factors associated with various physiological functions in plants which include cell division, secondary wall formation, formation of shoot apical meristem, leaf senescence, flowering embryo and seed development. Besides, they are also involved in biotic and abiotic stress regulation. Due to their ubiquitous influence, studies on NAC transcription factors have gained momentum in different crop plant species. In the present study, NAC25 like transcription factor was isolated and characterized from two cultivated coffee species, Coffea arabica and Coffea canephora and five Indian wild coffee species for the first time. The full-length NAC25 gene varied from 2,456 bp in Coffea jenkinsii to 2,493 bp in C. arabica. In all the seven coffee species, sequencing of the NAC25 gene revealed 3 exons and 2 introns. The NAC25 gene is characterized by a highly conserved 377 bp NAM domain (N-terminus) and a highly variable C terminus region. The sequence analysis revealed an average of one SNP per every 40.92 bp in the coding region and 37.7 bp in the intronic region. Further, the non-synonymous SNPs are 8-11 fold higher compared to synonymous SNPs in the non-coding and coding region of the NAC25 gene, respectively. The expression of NAC25 gene was studied in six different tissue types in C. canephora and higher expression levels were observed in leaf and flower tissues. Further, the relative expression of NAC25 in comparison with the GAPDH gene revealed four folds and eight folds increase in expression levels in green fruit and ripen fruit, respectively. The evolutionary relationship revealed the independent evolution of the NAC25 gene in coffee.

Genome-Wide Diversity Analysis of African Swine Fever Virus Based on a Curated Dataset.

African swine fever (ASF) is a lethal contagious viral disease of domestic pigs and wild boars caused by the African swine fever virus (ASFV). The pandemic spread of ASF has had serious effects on the global pig industry. Virus genome sequencing and comparison play an important role in tracking the outbreaks of the disease and tracing the transmission of the virus. Although more than 140 ASFV genome sequences have been deposited in the public databases, the genome-wide diversity of ASFV remains unclear. Here we prepared a curated dataset of ASFV genome sequences by filtering genomes with sequencing errors as well as duplicated genomes. A total of 123 ASFV genome sequences were included in the dataset, representing 10 genotypes collected between 1949 and 2020. Phylogenetic analysis based on whole-genome sequences provided high-resolution topology in differentiating closely related ASFV isolates, and drew new clues in the classification of some ASFV isolates. Genome-wide diversity of ASFV genomes was explored by pairwise sequence similarity comparison and ORF distribution comparison. Tandem repeat sequences were found widely distributed and highly varied in ASFV genomes. Structural variation and highly variable poly G or poly C tracts also contributed to the genome diversity. This study expanded our knowledge on the patterns of genetic diversity and evolution of ASFV, and provided valuable information for diagnosis improvement and vaccine development.

Evaluation of the HIV-1 Polymerase Gene Sequence Diversity for Prediction of Recent HIV-1 Infections Using Shannon Entropy Analysis.

HIV-1 incidence is an important parameter for assessing the impact of HIV-1 interventions. The aim of this study was to evaluate HIV-1 polymerase (pol) gene sequence diversity for the prediction of recent HIV-1 infections. Complete pol Sanger sequences obtained from 45 participants confirmed to have recent or chronic HIV-1 infection were used. Shannon entropy was calculated for amino acid (aa) sequences for the entire pol and for sliding windows consisting of 50 aa each. Entropy scores for the complete HIV-1 pol were significantly higher in chronic compared to recent HIV-1 infections (p < 0.0001) and the same pattern was observed for some sliding windows (p-values ranging from 0.011 to <0.001), leading to the identification of some aa mutations that could discriminate between recent and chronic infection. Different aa mutation groups were assessed for predicting recent infection and their performance ranged from 64.3% to 100% but had a high false recency rate (FRR), which was decreased to 19.4% when another amino acid mutation (M456) was included in the analysis. The pol-based molecular method identified in this study would not be ideal for use on its own due to high FRR; however, this method could be considered for complementing existing serological assays to further reduce FRR.

Characterization of the complete chloroplast genome sequences of six Dalbergia species and its comparative analysis in the subfamily of Papilionoideae (Fabaceae).

Dalbergia spp. are numerous and widely distributed in pantropical areas in Asia, Africa and America, and most of the species have important economic and ecological value as precious timber. In this study, we determined and characterized six complete chloroplast genomes of Dalbergia species (Dalbergia obtusifolia, D. hupeana, D. mimosoides, D. sissoo, D. hancei, D. balansae), which displayed the typical quadripartite structure of angiosperms. The sizes of the genomes ranged from 155,698 bp (D. hancei) to 156,419 bp (D. obtusifolia). The complete chloroplast genomes of Dalbergia include 37 tRNA genes, eight rRNA genes and 84 protein-coding genes. We analysed the sequence diversity of Dalberigia chloroplast genomes coupled with previous reports. The results showed 12 noncoding regions (rps16-accD, trnR-UCU-trnG-UCC, ndhE-ndhG, trnG-UCC-psbZ, rps8-rpl14, trnP-UGG-psaJ, ndhH-rps15, trnQ-UUG-rps16, trnS-GCU-psbI, rps12-clpP, psbA-trnK-UUU, trnK-UUU-intron), and four coding regions (rps16, ycf1, rps15 and ndhF) showed many nucleotide variations that could be used as potential molecular markers. Based on a site-specific model, we analysed the selective pressure of chloroplast genes in Dalbergia species. Twenty-two genes with positively selected sites were detected, involving the photosynthetic system (ndhC, adhD, ndhF, petB, psaA, psaB, psbB, psbC, psbK and rbcL), self-replication category of genes (rpoA, rpoC2, rps3, rps12 and rps18) and others (accD, ccsA, cemA, clpP, matK, ycf1 and ycf2). Additionally, we identified potential RNA editing sites that were relatively conserved in the genus Dalbergia. Furthermore, the comparative analysis of cp genomes of Dalbergieae species indicated that the boundary of IRs/SSC was highly variable, which resulted in the size variation of cp genomes. Finally, phylogenetic analysis showed an inferred phylogenetic tree of Papilionoideae species with high bootstrap support and suggested that Amorpheae was the sister of the clade Dalbergieae. Moreover, three genera of the Pterocarpus clade showed a nested evolutionary relationship. These complete cp genomes provided valuable information for understanding the genetic variation and phylogenetic relationship of Dalbergia species with their relatives.

Comparative pangenomics: analysis of 12 microbial pathogen pangenomes reveals conserved global structures of genetic and functional diversity.

BACKGROUND: With the exponential growth of publicly available genome sequences, pangenome analyses have provided increasingly complete pictures of genetic diversity for many microbial species. However, relatively few studies have scaled beyond single pangenomes to compare global genetic diversity both within and across different species. We present here several methods for "comparative pangenomics" that can be used to contextualize multi-pangenome scale genetic diversity with gene function for multiple species at multiple resolutions: pangenome shape, genes, sequence variants, and positions within variants. RESULTS: Applied to 12,676 genomes across 12 microbial pathogenic species, we observed several shared resolution-specific patterns of genetic diversity: First, pangenome openness is associated with species' phylogenetic placement. Second, relationships between gene function and frequency are conserved across species, with core genomes enriched for metabolic and ribosomal genes and accessory genomes for trafficking, secretion, and defense-associated genes. Third, genes in core genomes with the highest sequence diversity are functionally diverse. Finally, certain protein domains are consistently mutation enriched across multiple species, especially among aminoacyl-tRNA synthetases where the extent of a domain's mutation enrichment is strongly function-dependent. CONCLUSIONS: These results illustrate the value of each resolution at uncovering distinct aspects in the relationship between genetic and functional diversity across multiple species. With the continued growth of the number of sequenced genomes, these methods will reveal additional universal patterns of genetic diversity at the pangenome scale.

Genomic Analysis Unveils the Pervasiveness and Diversity of Prophages Infecting Erwinia Species.

Prophages are abundant elements integrated into bacterial genomes and contribute to inter-strain genetic variability and, in some cases, modulate the environmental behavior of bacteria, such as pathogen virulence. Here, we described prophage occurrence and diversity in publicly available Erwinia genome assemblies, a genus containing plant pathogens. Prophage-like sequences were identified and taxonomically classified. Sequence diversity was analyzed through intergenomic similarities. Furthermore, we searched for anti-phage defense systems in Erwinia spp., such as DISARM, BREX, and CRISPR-Cas systems, and identified the putative targets of CRISPR spacers. We identified 939 prophage-like sequences in 221 Erwinia spp. genome assemblies. Only 243 prophage-like sequences were classified, all belonging to the Caudoviricetes class. The set of putative Erwinia prophages was mostly unique since only three sequences showed more than 70% intergenomic similarities to known Erwinia phages. Overall, the number and type of CRISPR-Cas systems were conserved within Erwinia species, with many spacers directed to the putative prophages identified. This study increased the knowledge of the diversity and distribution of Erwinia prophages, contributing to the characterization of genetic and ecological factors influencing Erwinia spp. environmental fitness.

Non-homologous chromosome pairing: sequence similarity or genetic control?

Polyploids must correctly segregate homologous chromosomes. We propose that this process is dictated not just by sequence similarity, but is also under strong genetic control that may vary between lineages. We also highlight how factors like partner availability and genome structure may influence sequence similarity needed for crossover formation.

Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.

An Alignment-Independent Approach for the Study of Viral Sequence Diversity at Any Given Rank of Taxonomy Lineage.

The study of viral diversity is imperative in understanding sequence change and its implications for intervention strategies. The widely used alignment-dependent approaches to study viral diversity are limited in their utility as sequence dissimilarity increases, particularly when expanded to the genus or higher ranks of viral species lineage. Herein, we present an alignment-independent algorithm, implemented as a tool, UNIQmin, to determine the effective viral sequence diversity at any rank of the viral taxonomy lineage. This is done by performing an exhaustive search to generate the minimal set of sequences for a given viral non-redundant sequence dataset. The minimal set is comprised of the smallest possible number of unique sequences required to capture the diversity inherent in the complete set of overlapping k-mers encoded by all the unique sequences in the given dataset. Such dataset compression is possible through the removal of unique sequences, whose entire repertoire of overlapping k-mers can be represented by other sequences, thus rendering them redundant to the collective pool of sequence diversity. A significant reduction, namely ~44%, ~45%, and ~53%, was observed for all reported unique sequences of species Dengue virus, genus Flavivirus, and family Flaviviridae, respectively, while still capturing the entire repertoire of nonamer (9-mer) viral peptidome diversity present in the initial input dataset. The algorithm is scalable for big data as it was applied to ~2.2 million non-redundant sequences of all reported viruses. UNIQmin is open source and publicly available on GitHub. The concept of a minimal set is generic and, thus, potentially applicable to other pathogenic microorganisms of non-viral origin, such as bacteria.

Chloroplast Genome Sequencing, Comparative Analysis, and Discovery of Unique Cytoplasmic Variants in Pomegranate (Punica granatum L.).

Here we report on comprehensive chloroplast (cp) genome analysis of 16 pomegranate (Punica granatum L.) genotypes representing commercial cultivars, ornamental and wild types, through large-scale sequencing and assembling using next-generation sequencing (NGS) technology. Comparative genome analysis revealed that the size of cp genomes varied from 158,593 bp (in wild, "1201" and "1181") to 158,662 bp (cultivar, "Gul-e-Shah Red") among the genotypes, with characteristic quadripartite structures separated by a pair of inverted repeats (IRs). The higher conservation for the total number of coding and non-coding genes (rRNA and tRNA) and their sizes, and IRs (IR-A and IR-B) were observed across all the cp genomes. Interestingly, high variations were observed in sizes of large single copy (LSC, 88,976 to 89,044 bp) and small single copy (SSC, 18,682 to 18,684 bp) regions. Although, the structural organization of newly assembled cp genomes were comparable to that of previously reported cp genomes of pomegranate ("Helow," "Tunisia," and "Bhagawa"), the striking differences were observed with the Lagerstroemia lines, viz., Lagerstroemia intermedia (NC_0346620) and Lagerstroemia speciosa (NC_031414), which clearly confirmed previous findings. Furthermore, phylogenetic analysis also revealed that members outside the genus Punica were clubbed into a separate clade. The contraction and expansion analysis revealed that the structural variations in IRs, LSC, and SSC have significantly accounted for the evolution of cp genomes of Punica and L. intermedia over the periods. Microsatellite survey across cp genomes resulted in the identification of a total of 233 to 234 SSRs, with majority of them being mono- (A/T or C/G, 164-165 numbers), followed by di- (AT/AT or AG/CT, 54), tri- (6), tetra- (8), and pentanucleotides (1). Furthermore, the comparative structural variant analyses across cp genomes resulted in the identification of many varietal specific SNP/indel markers. In summary, our study has offered a successful development of large-scale cp genomics resources to leverage future genetic, taxonomical, and phylogenetic studies in pomegranate.

Reconstruction of Plasmodium vivax outbreaks in a low malaria endemic setting utilizing conventional restriction fragment length polymorphism.

Suriname is on track to eliminate local malaria transmission. P. vivax malaria reemerged in March and September 2019 in the Amerindian village Palumeu, free of malaria for two years and concurrently, a case was reported in another village Alalaparoe. The outbreaks were contained through targeted interventions including Mass Drug Administration (MDA). Molecular outbreak analysis was performed on 23 dried blood spots (DBS) using combined polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) with Pvmsp-1 F2 and Pvmsp-3α as polymorphic marker genes. Independent controls substantiated the discriminating capacities of the utilized PCR-RFLP method. All isolates from the first and second Palumeu outbreak shared a distinctive haplotype presuming single clonal lineage. An imported case probably triggered the first outbreak, while a delayed episode, prompted by withdrawal of drug pressure at the end of the prophylactic MDA, was suggested as source of the second outbreak. A diverging variant was demonstrated in Alalaparoe, implicating an infection from a different source. PCR-RFLP proved to be a useful molecular tool for P. vivax outbreak management in low endemic malaria settings.

Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis.

The optimal flowering time for bioenergy crop Miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in Miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8, a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis, we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species' subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A. The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.